The Ecology Investigation Of H7N9Subtype Influenza Virus And Study On Broad-spectrum Humanized Antibody For Anti-influenza Virus

In China, the case of H7N9influenza virus has occurred in February19,2013, and was confirmed inMarch. By the end of February in2014, the disease has caused more than350human cases, from Januaryto February2014, more than110people were infected,26people were fatal, Wild birds, especiallywaterfowl is considered the natural reservoir of H7N9The question is not well done for the H7N9influenza prevent and control. First all, the surveillance of H7N9in wild bird is limited in China. It isimportant to find out the characteristics ecology of H7N9virus for the prevention and control of H7N9inhuman. On the other hand, to develop an therapeutic agent is also important to the human infection cases.In order to reply to the two questions, we carried out two experiments including the investigation ofH7N9subtype of influenza virus in avian and broad-spectrum neutralization humanized antibodiesmanufacture.Experimental content and methods:1. The ecology investigation of H7N9subtype influenza virus2081feces samples of wild bird and poultries were collected from JiangSu, Jiangxi, Shanghai, Hunan,Jilin provinces and regions, virus isolation and subtypes identified were performed with SPF embryo andPCR. Analysis the H7N9subtype AIV of isolated by phylogenetic tree, the artificial infection, experimentsof domesticated mallard infected with influenza H7N9(Shanghai) virus assess. The results of the influenzaH7N9virus whether can spread and replicate.Experimental materials and methods:(1) virus isolation. Birds (waterfowl) and poultry samples wascollected and isolated influenza H7N9virus with9-day-old SPF embryo.(2) Virus subtype identification byRT-PCR and sequence analysis were performed for positive when AIV virus phylogenetic tree wasconstructed according the sequence.(3)The influenza H7N9virus receptor testing. The receptor binding ofH7N9virus was detected with SAα2-3Gal and SAα2-6Gal sugar. The reproduction capacities of H7N9virus in mallard were explore by experiment infection with A/Shanghai/2/2013H7N9virus.2. Study on broad-spectrum Humanized antibody for anti-influenza virus.Screening antibody sequence that can binding to the HA specific conserved regions of influenza viruswere conduct by using computer software to analysis the structure and function of the antibody that bindingto the HA region, the light and heavy chain variable region gene, through transformation, digestion, ligation,the light and heavy chain variable region gene connected the plasmids we constructed. Transfected intoinsect cells (sf9cells) with culture medium.Preliminary exploration for the broad-spectrum influenza virus humanized antibody. Experimental materials and methods:(1) through literature searches, protein sequence database and protein structuredatabase (PBD) accessed the sequence of antibody that screening. By computer analysis software InsightⅡ analysis antibody structure, through molecular modeling, molecular docking other methods, usinginsect cell preference codon optimized amino acid sequences, synthetic anti-influenza virus antibody lightand heavy chain variable region genes.(2) Construction of recombinant expression vector. The synthesisamino acid sequence of the light and heavy chain variable region gene was identified by restriction enzymedigestion, connected with PAC-κ-CH3, built PAC-κ-L-H-CH3recombinant expression vector, theidentification is correct, with the baculovirus DNA co-transfected into insect cells (sf9cells) and culture at27℃, about after4days use serum-free culture, then the supernatant was collected.(3) Identification forthe broad-spectrum. Humanized antibody. Using the inactivated influenza virus that store at our laboratory,to detect antibody Titer by hemagglutination inhibition test and ELISA, to detect antibodies in the cellexpression. by fluorescent antibody test.Results:1. The results of influenza H7N9virus ecology surveyFrom collected fecal samples of the wild bird, separated to the influenza H7N9virus, but the resultswas H7subtypes positive in the serum and eggs by hemagglutination inhibition test.We isolated and identified two influenza H7N9viruses from the poultry samples of Changchun byRT-PCR and SPF chicken embryos. One of sample, which show H7N9virus positive, were H9N2avianinfluenza virus positive.HA and NA full-length sequences was determined, using BLAST of NCBI website, and MAGE6.0software built phylogenetic tree. The HA and NA phylogenetic analysis showed that the influenza virusisolated from this experiment have the correlation is98.6%and99.2%with the influenza H7N9virusisolated from Guangzhou and Fujian province.The influenza H7N9virus of isolates recognize both SAα2-3Gal and SAα2-6Gal receptor, and thevirus binding ability to SAα2-3Gal is stronger than it to SAα2-6Gal.In mallard challenged experiments, the same group and the control group, Mallard had not any clinicalsymptoms, and the virus had not been detected from the collected samples from the mallard. The resultsindicated that the influenza virus (A/Shanghai/2/2013, H7N9) can not replicate in the mallard.2. Preliminary exploration for the broad-spectrum influenza virus humanized antibodyWe find6anti-influenza antibody sequences with Literature search protein sequence databases,Protein Structure Database (PBD), the antibody sequences was been analyzed by computer with softwareInsight Ⅱ.The antibody sequences were named A66, H40, H98, F10, JAN, D80. The antibody of F10canbinding with HA1antibody recognition from the diagram.The recombinant baculovirus expression vector was identified correctly. Light and heavy chain variable region gene is properly connected to the vector.Through the transfected of the recombinant baculovirus express vector into cell sf9, obtain3antibodythat broad-spectrum inhibition different influenza subtype virus (H1N1, H5N1and H9N2), named H40,H98and A66. antibody can against two influenza subtype virus, and F10against H7N9and H1N1,JANneutralized H1N1and H5N1, The other is no neutralizing to influenza virus. There is positive expressed byfluorescent antibody test.Conclusion1. We isolated2H7N9influenza virus from the2081samples.The influenza H7N9virus can bindsα2-3and α2-6sciatic acid receptors, Phylogenetic analysis indicated that the influenza H7N9virus at thesame branch with H7N9virus from Guangzhou and Fujian province with the correlation98.6%and99.2%.The results were confirm with artificially infected mallards with A/Shanghai/2/2013(H7N9) virusstrains and the virus cannot replicate and spread in the mallard’s body.2. The humanized antibody show broad-spectrum specificity for different subtype virus: H40, H98and A66. Neutralizing different influenza subtype virus H40, H98and A66. F10could against H7N9andH1N1virus against to H5N1and H1N1.In conclusion, our study provided preliminary inference for the ecology H7N9influenza virus. H7N9virus is not exist in wild birds, the infection was confirmed in experiments of artificial mallard withinfluenza H7N9A viruses.However, the H7subtype was been detected in serum and egg yolk, it also showsthat the risk is exit that wild birds maybe exist other influenza subtypes. Preliminary exploration for thebroad-spectrum influenza virus humanized antibody. Three humanized antibodies for influenza virus wereselected and indented field base on the sequences from references. However, we found that any antibodycould not be reaction with all influenza A virus. Indicating that the antibody treatment diseases in clinicaluse mix monoclonal antibody is better choice. Our study provides a basic database for the development ofhumanized antibody.